The purpose of this project is to determine the mechanism by which senescent human red blood cells (RBC) and RBC stored in vitro for transfusion are removed from the circulation. Previous experiments performed in this laboratory suggest that macrophages distinguish autologous senescent from mature RBC on the basis of selective IgG attachment to the former. In the proposed studies, the IgG will be eluted from senescent cells and RBC aged in vitro, quantitated and characterized by immunochemical, biochemical, and biological assays to determine whether it meets the criteria of an autoantibody. The eluted IgG will be tested for binding and specificity using 2 complementary assays: (a) a scanning immunoelectron microscopy (SIEM) labeling technique which enables detection of as few as 2 Ig molecules per cell and assesses density and distribution of receptor sites for many molecules, and (b) a phagocytosis assay which assesses recognition and ingestion of RBC by macrophages. If the IgG which binds the senescent RBC in situ proves to be an autoantibody, this would establish the existence of physiologic autoantibodies. The IgG-binding receptor on RBC will then be identified immunochemically. These studies may reveal methods for: (a) devising a simple in vitro method of predicting the transfusion effectiveness of stored blood, (b) devising a simple method of removing nonsenescent RBC from stored blood which otherwise would be discarded, (c) devising a better storage medium for RBC, and (d) altering the receptor so that the half life of stored cells can be prolonged -- methods which would improve the Blood Bank program. The results of experiments performed in vitro will be confirmed in situ using an animal model and 59Fe labeled RBC.